PO.MCB04.02 · 分子与细胞生物学
线粒体谷胱甘肽还原酶作为KEAP1/NRF2突变型非小细胞肺癌的氧化还原易损性
Mitochondrial glutathione reductase as a redox vulnerability in KEAP1/NRF2-mutant non-small cell lung cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
非小细胞肺癌（NSCLC）占肺癌的85-90%。NRF2是一种协调抗氧化防御的主转录因子。在基础条件下，KEAP1结合NRF2，促进其泛素化和降解。氧化应激改变KEAP1的构象，阻止其与NRF2结合，从而使NRF2在核内累积并激活细胞保护性基因。NSCLC常携带KEAP1或NRF2突变——通常位于KEAP1的Kelch结构域或NRF2的Neh2结构域内——这些突变损害KEAP1介导的周转并驱动NRF2的组成型激活。此类肿瘤对化疗、放疗和免疫治疗具有抵抗性，然而由于缺乏催化口袋以及对系统性毒性的担忧，直接的NRF2抑制剂仍然难以获得。为了鉴定能够增强NRF2活跃型NSCLC中ROS诱导性死亡的合成致死靶点，我们使用一个聚焦的抗氧化酶sgRNA文库进行了负选择CRISPR/Cas9筛选。细胞用亚致死剂量的NQO1可生物激活的ROS生成前药β-拉帕醌（beta-lapachone）处理。正如预期，NQO1是最主要的致敏命中基因，而TXNRD1/TXN在抵抗性命中中富集，这与硫氧还蛋白通路的保护作用一致。出乎意料的是，从头合成谷胱甘肽所需的基因（GCLM、GCLC、GSS）显示出极小的脱落，表明GSH生物合成对β-Lap敏感性的贡献有限。相反，负责将GSSG还原为GSH的NRF2靶基因GSR显著凸显出来，提示GSR缺失在氧化应激下独特地损害了细胞存活。在多种ROS诱导剂中，GSR敲除强烈致敏了KEAP1/NRF2突变型细胞，但对KEAP1/NRF2完整型细胞无此作用。重要的是，其毒性并非由全局性GSH耗竭所驱动。相反，我们发现线粒体GSR是必需的：仅有胞质定位的GSR无法挽救GSR缺陷细胞，而强制线粒体定位则完全恢复了细胞活力。在机制上，GSR缺失导致严重的线粒体碎裂，损害了电子传递链（ETC）的完整性，并降低了ETC活性。GSR缺陷细胞积累线粒体ROS，并表现出含铁硫簇蛋白表达的降低，共同驱动线粒体功能障碍和细胞死亡。在体内，GSR缺失触发了氧化还原崩溃，并显著地使KEAP1突变型肿瘤对化疗致敏。总之，这些发现揭示了KEAP1/NRF2突变型NSCLC中一种此前未被认识的线粒体氧化还原依赖性，并将GSR确定为克服NRF2介导的ROS抵抗的一个有前景的治疗易损性。
查看英文原文 English abstract
Non-small cell lung cancer (NSCLC) accounts for 85-90% of lung cancers. NRF2 is a master transcription factor that orchestrates antioxidant defense. Under basal conditions, KEAP1 binds NRF2, promoting its ubiquitination and degradation. Oxidative stress alters KEAP1 conformation, preventing NRF2 binding and enabling NRF2 nuclear accumulation and activation of cytoprotective genes. NSCLC frequently harbors KEAP1 or NRF2 mutations-often within the KEAP1 Kelch domain or the NRF2 Neh2 domain-that impair KEAP1-mediated turnover and drive constitutive NRF2 activation. Such tumors are resistant to chemo-, radio-, and immunotherapy, yet direct NRF2 inhibitors remain elusive due to the absence of catalytic pockets and concerns about systemic toxicity. To identify synthetic-lethal targets that heighten ROS-induced death in NRF2-active NSCLC, we performed negative-selection CRISPR/Cas9 screens using a focused antioxidant-enzyme sgRNA library. Cells were treated with a sublethal dose of the NQO1-bioactivatable ROS-generating prodrug beta-lapachone. As expected, NQO1 was the top sensitizing hit, and TXNRD1/TXN were enriched among resistant hits, consistent with the protective thioredoxin pathway. Unexpectedly, genes required for de novo glutathione synthesis (GCLM, GCLC, GSS) showed minimal dropout, indicating limited contribution of GSH biosynthesis to beta-Lap sensitivity. Instead, the NRF2 target gene GSR-responsible for reducing GSSG to GSH-emerged prominently, suggesting that GSR loss uniquely impairs survival under oxidative stress. Across multiple ROS-inducing agents, GSR knockout strongly sensitized KEAP1/NRF2-mutant cells but not KEAP1/NRF2-intact cells. Importantly, toxicity was not driven by global GSH depletion. Rather, we found that mitochondrial GSR is essential: cytosolic-only GSR could not rescue GSR-deficient cells, whereas forced mitochondrial localization fully restored viability. Mechanistically, GSR loss caused severe mitochondrial fragmentation, compromised electron transport chain (ETC) integrity, and reduced ETC activities. GSR-deficient cells accumulated mitochondrial ROS and showed decreased expression of iron-sulfur cluster-containing proteins, collectively driving mitochondrial dysfunction and cell death. In vivo, GSR deletion triggered redox collapse and dramatically sensitized KEAP1-mutant tumors to chemotherapy. Together, these findings reveal an unrecognized mitochondrial redox dependency in KEAP1/NRF2-mutant NSCLC and identify GSR as a promising therapeutic vulnerability for overcoming NRF2-mediated ROS resistance.
利益披露 Disclosure
C. Ting, None..
J. Lin, None..
C. Jiang, None.